title: Novel, gel-free proteomics approach identifies RNF5 and JAMP as modulators of GPCR stability.
abstract: The maturation and folding of G protein-coupled receptors are governed by mechanisms that remain poorly understood. In an effort to characterize these biological events, we optimized a novel, gel-free proteomic approach to identify partners of the β2-adrenergic receptor (β2AR). In addition to a number of known interacting proteins such as heterotrimeric G protein subunits, this allowed us to identify proteins involved in endoplasmic reticulum (ER) QC of the receptor. Among β2AR-associated proteins is Ring finger protein 5 (RNF5), an E3 ubiquitin ligase anchored to the outer membrane of the ER. Coimmunoprecipitation assays confirmed, in a cellular context, the interaction between RNF5 and the β2AR as well as the prostaglandin D2 receptor (DP). Confocal microscopy revealed that DP colocalized with RNF5 at the ER. Coexpression of RNF5 with either receptor increased levels of their expression, whereas small interfering RNA-mediated knockdown of endogenous RNF5 promoted the opposite. RNF5 did not modulate the ubiquitination state of β2AR or DP. Instead, RNF5 ubiquitinated JNK-associated membrane protein (JAMP), a protein that recruits the proteasome to the ER membrane and that is negatively regulated by RNF5-mediated ubiquitination. JAMP coimmunoprecipitated with both β2AR and DP and decreased total receptor protein levels through proteasomal degradation. Expression of DP, a receptor largely retained in the ER, promoted proteasome recruitment by JAMP. Degradation of both receptors via JAMP was increased when RNF5 was depleted. Our data suggest that RNF5 regulates the turnover of specific G protein-coupled receptors by ubiquitinating JAMP and preventing proteasome recruitment.
authors.affiliation: Service de Rhumatologie Département de Médecine, Université de Sherbrooke, the Institut de Pharmacologie de Sherbrooke, and the Centre de Recherche Clinique Etienne-Lebel, Sherbrooke, Québec J1H 5N4, Canada.
authors.forename: Sébastien J
authors.initials: SJ
authors.lastname: Roy
authors.forename: Irina
authors.initials: I
authors.lastname: Glazkova
authors.forename: Louis
authors.initials: L
authors.lastname: Fréchette
authors.forename: Christian
authors.initials: C
authors.lastname: Iorio-Morin
authors.forename: Chantal
authors.initials: C
authors.lastname: Binda
authors.forename: Darlaine
authors.initials: D
authors.lastname: Pétrin
authors.forename: Phan
authors.initials: P
authors.lastname: Trieu
authors.forename: Mélanie
authors.initials: M
authors.lastname: Robitaille
authors.forename: Stéphane
authors.initials: S
authors.lastname: Angers
authors.forename: Terence E
authors.initials: TE
authors.lastname: Hébert
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Parent
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D021381:Protein Transport; D040901:Proteomics; D034622:RNA Interference; D034741:RNA, Small Interfering; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000378:metabolism*; D011982:Receptors, Prostaglandin / Q000378:metabolism*; D015398:Signal Transduction; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C541074:JKAMP protein, human; D008562:Membrane Glycoproteins; D034741:RNA, Small Interfering; D018343:Receptors, Adrenergic, beta-2; D011971:Receptors, Immunologic; D011982:Receptors, Prostaglandin; C111501:RNF5 protein, human; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; C036293:prostaglandin D2 receptor